Abstract

Background

As well as being inducible by haem, haemoxygenase -1 (HO-1) is also induced by interleukin-10
and an anti-inflammatory prostaglandin, 15d PGJ2, the carbon monoxide thus produced mediating the anti-inflammatory effects of these
molecules. The cellular distribution of HO-1, by immunohistochemistry, in brain, lung
and liver in fatal falciparum malaria, and in sepsis, is reported.

Methods

Wax sections were stained, at a 1:1000 dilution of primary antibody, for HO-1 in tissues
collected during paediatric autopsies in Blantyre, Malawi. These comprised 37 acutely
ill comatose patients, 32 of whom were diagnosed clinically as cerebral malaria and
the other 5 as bacterial diseases with coma. Another 3 died unexpectedly from an alert
state. Other control tissues were from Australian adults.

Results

Apart from its presence in splenic red pulp macrophages and microhaemorrhages, staining
for HO-1 was confined to intravascular monocytes and certain tissue macrophages. Of
the 32 clinically diagnosed cerebral malaria cases, 11 (category A) cases had negligible
histological change in the brain and absence of or scanty intravascular sequestration
of parasitized erythrocytes. Of these 11 cases, eight proved at autopsy to have other
pathological changes as well, and none of these eight showed HO-1 staining within
the brain apart from isolated moderate staining in one case. Two of the three without
another pathological diagnosis showed moderate staining of scattered monocytes in
brain vessels. Six of these 11 (category A) cases exhibited strong lung staining,
and the Kupffer cells of nine of them were intensely stained. Of the seven (category
B) cases with no histological changes in the brain, but appreciable sequestered parasitised
erythrocytes present, one was without staining, and the other six showed strongly
staining, rare or scattered monocytes in cerebral vessels. All six lung sections not
obscured by neutrophils showed strong staining of monocytes and alveolar macrophages,
and all six available liver sections showed moderate or strong staining of Kupffer
cells. Of the 14 (category C) cases, in which brains showed micro-haemorrhages and
intravascular mononuclear cell accumulations, plus sequestered parasitised erythrocytes,
all exhibited strong monocyte HO-1 staining in cells forming accumulations and scattered
singly within cerebral blood vessels. Eleven of the available and readable 13 lung
sections showed strongly staining monocytes and alveolar macrophages, and one stained
moderately. All of the 14 livers had strongly stained Kupffer cells. Of five cases
of comatose culture-defined bacterial infection, three showed a scattering of stained
monocytes in vessels within the brain parenchyma, three had stained cells in lung
sections, and all five demonstrated moderately or strongly staining Kupffer cells.
Brain sections from all three African controls, lung sections from two of them, and
liver from one, showed no staining for HO-1, and other control lung and liver sections
showed few, palely stained cells only. Australian-origin adult brains exhibited no
staining, whether the patients had died from coronary artery disease or from non-infectious,
non-cerebral conditions

Conclusions

Clinically diagnosed 'cerebral malaria' in children includes some cases in whom malaria
is not the only diagnosis with the hindsight afforded by autopsy. In these patients
there is widespread systemic inflammation, judged by HO-1 induction, at the time of
death, but minimal intracerebral inflammation. In other cases with no pathological
diagnosis except malaria, there is evidence of widespread inflammatory responses both
in the brain and in other major organs. The relative contributions of intracerebral
and systemic host inflammatory responses in the pathogenesis of coma and death in
malaria deserve further investigation.

Introduction

Falciparum malaria is a complex multi-organ disease. There is no simple or accepted
explanation for how small numbers of parasites can cause such severe illness, or how
this infection can cause such wide-spread pathology, since only hepatocytes and erythrocytes
are invaded by the pathogen. Undoubtedly parasitized red cells sequester in capillaries
and venules, but in recent times the traditional idea that this is the primary cause
of organ failure and death through obstructing blood flow has needed modifying. In
particular, it has had to accommodate the evident involvement of excessive systemic
release of pro-inflammatory cytokines, triggered by malarial toxins. For the last
decade, many researchers have focussed their efforts on the pathophysiological implications
of the ability of these mediators to generate inducible nitric oxide synthase (iNOS),
and thus produce a continuous, potentially large, supply of nitric oxide in tissues
that normally experience only low, tightly controlled, levels of this ubiquitous cellular
messenger. Despite the harmful effects of iNOS-induced nitric oxide (NO) when produced
in unusually large amounts [1-3], more commonly it provides negative feedback that suppresses production of the inflammatory
cytokines that generate it, and a range of other downstream harmful mediators, through
inhibiting NF kappa B, a major activator of protein transcription [4].

Carbon monoxide (CO), another endogenous gas with a similar structure, also inhibits
TNF generation [5] again through inhibiting NF kappa B [6]. Both molecules are generated by enzymes that have at least one constitutive form,
and another, iNOS and haemoxygenase-1 (HO-1) respectively, induced by inflammatory
cytokines. NO and CO act interactively as second messengers in ways that are still
being elucidated [7]. For instance, both NO and CO can activate soluble guanylate cyclase to generate
cyclic GMP [8], and thus dilate blood vessel walls, as well as perform their immunosuppressive roles.

This shared activity of NO and CO duplicates that of interleukin-10 (IL-10), the prototype
anti-inflammatory cytokine, which also suppresses generation of tumour necrosis factor
(TNF) and interleukin-1β through inhibiting NF kappa B [9]. Thus the high circulating levels of IL-10 seen in human malaria [10,11] and sepsis [12], have been proposed to suppress disease severity through inhibiting the systemic
inflammatory effects of TNF [13]. These apparently disparate observations are now appreciated to be different parts
of the same chain of events, with IL-10 producing its strong anti-TNF effect through
inducing HO-1, and thus generating CO [14]. The most plausible explanation for the early observation that TNF induces HO-1 [15] is now therefore its ability to induce IL-10 [16]. Likewise, the anti-inflammatory effect of 15d prostaglandin J2 (15d PGJ2), which is present in tissues during inflammation [17], also operates through HO-1 induction and subsequent generation of CO [18]. As in sepsis, cyclooxygenase-2, which generates 15d PGJ2, is induced in severe malaria [19]. Thus, rather than HO-1 simply being a marker for haem degradation and a generator
of anti-oxidant defences, it is now recognized to be an integral part of the network
of inflammatory mediators. It therefore serves as a convenient and sensitive marker
for such activity.

Accordingly, brain, lung and liver from 40 African children who had died of malaria,
sepsis or unrelated conditions were stained for HO-1 in order to identify cellular
sites where the CO-mediated anti-inflammatory activity of IL-10 might be located in
these infectious diseases. These tissues had previously been stained for migration
inhibitory factor (MIF) and inducible nitric oxide synthase (iNOS) [20]. Before the IL-10 or prostaglandin links of HO-1 were appreciated, others [21,22] have immunostained brains, but no other tissues, from adult malaria cases, to detect
this enzyme. The present study provides further evidence for the presence of multi-organ
inflammatory changes in children fulfilling the clinical criteria of 'cerebral malaria',
whether or not malaria was the principal or only pathological diagnosis.

Materials and Methods

Case Tissues

As described earlier [20], all 40 subjects (age range six months to 12 years; 22 females) were children who
had been admitted to the Malaria Project wards in the Department of Paediatrics at
the Queen Elizabeth Central Hospital in Blantyre, Malawi (Table). Evaluation, diagnoses,
treatment, autopsy permissions were as previously described [20]. Autopsies were performed as quickly after death as possible, with post-mortem intervals
ranging from two to 14.5 hrs. Tissue samples were placed into 10% neutral buffered
formalin for fixation. The project was approved by the ethics and research boards
of the College of Medicine (University of Malawi), the University of Liverpool and
the Australian National University.

Control tissues

Tissues from three Malawian children who were enrolled in this study served as local
non-comatose controls. No coma was present at any stage in two of these (patients
41 and 50; see Table). The former grew Salmonella typhimurium from cerebrospinal fluid and blood, and died, having been alert a short time before,
after an acute gastrointestinal haemorrhage, and the other grew scanty Streptococcus pneumoniae from the cerebrospinal fluid. The third (patient 43) had been diagnosed as cerebral
malaria but, after recovering to an alert state, died from a cardiopulmonary arrest.
In addition, various adult controls from Australian sources were studied. These comprised
sections of five blocks of tissue, trimmed from the periphery of tumour excisions
from adult chest wall, and containing skeletal muscle, adipose tissue and small blood
vessels. Midbrain sections from three adults who had died of coronary artery disease,
and from another three who died of non-infectious, non-cerebral conditions (Brain
Bank for Sydney Central Area Health Science Approval X980216), were also stained.
A section of an inflamed pilonidal sinus was routinely included as a positive control.

Immunohistochemistry

Formalin-fixed tissue samples were embedded in paraffin, sectioned (4 microns) on
to polylysine-coated slides, and stained with haematoxylin and eosin (H&E) for routine
morphology. A monoclonal anti-HO-1 antibody was purchased from StressGen; (Cat. No.
OSA-110). Other monoclonals were used as irrelevant primary control antibodies, and
in other controls the primary antibody was omitted. As previously [20], antigen retrieval was performed by immersion in 0.01 M citrate buffer, pH 6.0, in
a waterbath at 95°C for 20 min and then cooling to room temperature while still immersed
in buffer. After quenching with 3% H2O2 and treating with primary antibody (dilution of the stock solution 1:500 to 1:2000)
at room temperature for 1 hr, biotin-conjugated secondary antibody and streptavidin-conjugated
horseradish peroxidase from an LSAB+ kit (DAKO) were applied to sections for 20 min at room temperature to amplify the
antigen signal for subsequent 3,3'-diaminobenzidine (DAB) staining. Known positive
controls were stained in each run, and runs were often duplicated on different days
to confirm repeatability. Sections were counterstained with haematoxylin, and outcomes
with a dilution of primary antibody of 1:1000 are shown to illustrate the observed
changes. Anti-CD68 antibody (Clone PG-M1) was obtained from DAKO, and used, with antigen
retrieval, at a primary antibody dilution of 1:500.

Histological examination

In a recent investigation of the distribution of MIF and iNOS [20], in which 32 cases that had been clinically diagnosed as cerebral malaria were studied,
they were classified into three categories on the basis of the presence or absence
of sequestered intracerebral parasites and brain pathology. Category A (n = 11) had
no or scanty intracerebral parasites and negligible brain pathology detected, category
B (n=seven) had sequestered parasites in brain vessels, again with negligible brain
pathology, and category C (n = 14) had both sequestered parasites and inflammatory
brain pathology in the form of intravascular monocyte aggregations, fibrin deposition
and/or microhaemorrhages. Here this same terminology is retained, and the outcome
of immunostaining to detect HO-1 in the brain, lung and liver of these same cases
is reported. In most of the category A patients, autopsy revealed another likely cause
of death: this was pneumonia in five cases, hepatic necrosis in one, severe anaemia
with pulmonary oedema in one, and ruptured cerebral aneurysm in one. No alternative
causes were identified in category B and C patients.

One hundred and forty sections from 49 brains were stained for HO-1 and examined.
Samples were from frontal lobe, parietal lobe, temporal lobe, occipital calcarine
fissure, hippocampus, caudate nucleus, basal ganglia, thalamus, midbrain, pons and
medulla, with frontal lobe, occipital region, midbrain and pons most commonly included.
Two sections were considered ample to record a result on sections where staining was
readily detected, since up to seven were examined in some cases, with no difference
in outcome from the opinion formed after the first section. Up to seven sections per
brain were examined in those cases with no staining detectable, and these comprised
sections from two to four blocks, which provided an element of depth within individual
blocks as well as a spread of location across the brain. With few exceptions, staining
was either absent, moderate, or strong, and if strong was sometimes remarkably intense
and even, despite the high dilution of primary antibody. Mid-brain sections from three
adults who had died of coronary artery disease, and from another three who died of
non-infectious, non-cerebral conditions (Brain Bank for Sydney Central Area Health
Science Approval X980216), were included. Single blocks only were available for lung
and liver. Two examiners (IC and CH), blinded to the diagnosis, examined the sections
independently.

Results

Cerebral malaria

Brain of the 32 cases clinically diagnosed as cerebral malaria were examined after
staining with anti-HO-1 antibody as described. Differences in degree of staining between
the various parts of the brain in any single individual were slight to negligible,
with the picture observed in the first section examined holding true throughout the
series for that case. Staining was restricted to monocytes, and where microhaemorrhages
were present HO-1 was detected in the macrophages that migrated into the immediate
area. In contrast to iNOS staining, a characteristic of HO-1 detection was its either
strong presence or absence, with little between. Only single blocks of lung and liver
were available, and these stained uniformly across the section. Staining in lung was
restricted to monocytes and alveolar macrophages, and in liver to Kupffer cells, except
for a very pale even stain across the parenchyma of a few livers.

Category A cerebral malaria

In the 11 cases that comprised category A (no discernible histological changes, only
the occasional rare malarial pigment or parasites within vessels, and in most cases
an additional explanation for death identified at general autopsy) eight had no detectable
HO-1 staining. in the brain (Table 1). One showed isolated moderately stained monocytes, and the other two showed a light
scattering of strongly stained monocytes across the brain parenchyma. Examples are
shown in Fig. 1. The identity of these cells was confirmed with CD68 staining (not shown). Of the
11 lung sections, six exhibited strongly stained monocytes and alveolar macrophages
(Fig. 1), two were pale, and one was negative. In the other two the section was largely obscured
by a heavy influx of neutrophils, a cell type we have not seen stain for HO-1. Nine
of the 11 liver sections showed strongly staining Kupffer cells (Fig. 1), and in the other two these cells were moderately stained only.

Figure 1.CM (A). HO-1 staining of tissues from cerebral malaria cases showing apparently absent
physical brain pathology. A and B brain, C and D lung, and E and F liver, the left hand column (A, C and E) being from case 22, an example of the more
common (8 of 11) combination, in which cerebral vasculature monocytes were quiescent.
B, D and F are from case 38. As in both examples, monocytes in lung, and Kupffer cells,
were commonly (6 of 11 and 9 of 11 respectively) strongly stained. Scale bar, 100
μm.

Category B cerebral malaria

In the seven category B cases (no discernible pathological changes in the examined
sections, but sequestered parasitised erythrocytes present) 2 had rare strongly staining
monocytes in cerebral blood vessels, and another four showed a scattering of strongly
stained monocytes (Table 1). The identity of these cells was confirmed with CD68 staining (not shown) An example
of each type of density is shown in Fig. 2. Five out of seven lung sections showed strong staining of monocytes and alveolar
macrophages, one was moderately stained, and the other section was largely obscured
by a heavy influx of neutrophils, with no HO-1 staining discernible. All six of the
available seven liver sections showed strong staining of Kupffer cells, again as illustrated
in Fig. 2.

Figure 2.CM (B). HO-1 staining of tissues from cerebral malaria cases showing apparently absent
physical brain pathology, but sequestered parasites common. A and B brain, C and D lung, and E and F liver. The left hand column (A, C and E) is from an example (case 25) of the more
common (4 of 7) combination, in which cerebral vasculature monocytes were not common,
though parasite sequestration sometimes intense. B, D and F are from case 21. As in
both examples, monocytes in lung, and Kupffer cells, were commonly strongly stained.
Scale bar, 100 μm.

Category C cerebral malaria

The brains of 14 category C cases (microhaemorrhages, intravascular mononuclear cell
accumulations, and also sequestered parasites) all exhibited strong monocyte HO-1
staining in mononuclear leucocytes forming accumulations, and also in those scattered
singly, within blood vessels (Table 1). The two cases illustrated in Fig. 3 had neither a low haematocrit, nor hypoglycaemia. Monocyte accumulations were not
seen in sections of two of the brains, and thus the individual cells are referred
to in the Table as "scattered, strong". In addition the macrophages that had aggregated
at microhaemorrhages were stained strongly (Fig. 3). The identity of these cells was confirmed with CD68 staining (not shown). Eleven
of the 13 available lung sections showed strongly staining monocytes and alveolar
macrophages (Fig. 3), with the monocytes often in accumulations similar to those observed in the brain.
One section was moderately stained, and the other section was largely obscured by
a heavy influx of neutrophils. As shown in the examples in Fig. 3, all of the 14 livers had strongly stained Kupffer cells.

Coma associated with bacterial infections

Another five patients were comatose on admission and before death, had very low or
no peripheral malaria parasitaemia, and were considered on clinical grounds to be
suffering from a disease other than malaria [20]. Two had septic meningitis (one each culture positive for Haemophilus influenzae and S. pneumoniae), two had a positive blood culture (one Salmonella enteritidis, one Escherichia coli) and one, with a lymphocytic infiltrate in the cerebrospinal fluid, was diagnosed
histologically, including the presence of acid-fast organisms, as tuberculous meningitis.
The E. coli and H. influenzae cases had no detectable HO-1 staining. in the brain, and the other three showed a
variable scattering of stained monocytes (Table 1). In the tuberculosis case staining was limited to the areas adjacent to large blood
vessels. Two of the five lung sections (S. enteritidis and E. coli cases) showed strongly staining monocytes and alveolar macrophages, while none were
evident in the H. influenzae and S. pneumonia cases, and they were restricted to the region near chronic lesions in the tuberculosis
case. All five livers showed HO-1 staining of the Kupffer cells, although in one case
this was moderate rather than strong (Table 1). As examples, the changes in brain, lung and liver of the E. coli and H. influenzae cases are shown in Fig. 4.

Control Tissues

Brain sections from all three African controls, and lung sections from two of them,
and the liver of one, showed no staining for HO-1 (Table 1, Fig. 5). The lung of the one, and the liver of two, showed few, palely stained cells only.
Likewise, the 6 Australian-origin adult brains showed no staining, whether the patients
had died from coronary artery disease or non-infectious, non-cerebral conditions (Fig.
5G). The subcutaneous tissues from Australian adult controls were also negative (not
shown). In contrast, macrophages in the section of inflamed pilonidal sinus included
in every staining run invariably were strongly positive (Fig. 5H), as were the macrophages in the splenic red pulp, a location where free haem can
be expected to induce HO-1, in all African cases (not shown).

Figure 5.HO-1 staining of tissues from controls. A and B brain, C and D lung, and E and F liver of African controls, cases 41 (A, C and E) and 43 (B, D and F). Australian origin
coronary artery disease brain (G) and chronically infected pilonidal sinus (H) are also shown. Pattern shown was the same in all cases illustrated. Scale bar,
100 μm.

CD 68 staining

CD36 staining confirmed the identity of the mononuclear phagocytic lineage of stained
cells, including the cells attracted to ring haemorrhages, that were positive for
HO-1. Both single and double staining was undertaken (not shown). This confirmed that
negative HO-1 staining of tissues could not be attributed to an absolute absence of
monocytes and macrophages in various locations, but to them being present and not
staining.

Discussion

Here is described the cellular distribution of HO-1 in several key organs in African
children who died of clinically defined cerebral malaria or coma accompanying a bacterial
infection. Endothelial cells and vascular smooth muscle, and skeletal muscle, so often
MIF and iNOS positive [20], were devoid of HO-1. Previous reports of HO-1 staining of human tissues includes
the cytotrophoblast cells within the placental bed of the normal human placenta [23], the alveolar macrophages of normal and inflamed lung [24], endothelium and macrophages of atherosclerosis lesions [25], Kupffer cells and hepatocytes in portal hypertension [26], tubular epithelial cells in kidney diseases [27], and microglia and macrophages in focal cerebral infarcts and brain trauma [28]. Malarial brains, but no other organ, from Asian [22] and European adults [21] have been stained for HO-1, and, consistent with the literature of the time, HO-1
was discussed only in terms of being a stress protein [22] and a generator of CO that could contribute to cerebral malaria by influencing neurons
directly [21].

What induced HO-1 in these tissues remains unclear. It is well accepted that a high
concentration of haem induces HO-1 [29], and the invariable staining observed in spleen red pulp in all cases (not shown),
and at cerebral haemorrhage sites where red cells have been phagocytosed (Fig. 3B) in category C brains is consistent with this. Haem from local haemorrhage cannot,
however, account for the presence of HO-1 in mononuclear phagocytes in CM(A) or CM(B)
brains, and other organs In addition, cellular deprivation of glucose has been shown
to induce the HO-gene 1 in vitro [30], raising the possibility of the hypoglycaemia sometimes seen in severe childhood
falciparum malaria being instrumental in generating the observed HO-1. No difference
was seen in the intensity of HO-1 staining between cases with normal peripheral blood
glucose concentration and those with systemic hypoglycaemia, but this observation
does not exclude local tissue hypoglycaemia as a possible stimulus to HO-1 induction.
IL-10, a major modulator of inflammation increased in malaria [10], is now known to induce HO-1 [14]. Plasmas for IL-10 assay were not available from these cases, but the precedence
exists of high levels being associated with severe malarial illness [10,11].

In view of the evidence that the inflammatory cascade is activated in this Malawian
[20,31] and other [32-34] populations with severe malaria infection, the observed HO-1 is additional evidence
that in fatal malaria a widespread host inflammatory response occurs, similar to that
seen in other acute infections (reviewed in [35]). This is consistent with the HO-1 staining seen in the sepsis cases in this series
(Fig. 4), in that systemic bacterial infections are broadly accepted to be examples of systemic
inflammation. This is not to suggest that some of the cerebral malaria cases in this
series did not have additional or alternative disease mechanisms, arising from local
cerebral lesions, such as vessel obstruction from large parasite loads in the apparent
absence of other lesions (Fig. 2B). Nevertheless, strong systemic inflammation was present in all these cases. Local
cerebral changes arising from post-schizogony secondary inflammatory events, as demonstrated
by the presence of monocytes accumulations, high iNOS, haemorrhages and fibrin in
other CM(C) cases (Fig. 3A and 3B) may well also contribute significantly.

Taken with the evidence that eight of the 11 category A brains had no evidence of
HO-1 staining, these results, along with the apparent absence of sequestration or
monocytic accumulation (now reinforced by examining this large series of new sections)
and iNOS staining in these same brains [20], add to previous evidence [20] that these 11 sets of brains sections, one third of the total from clinically diagnosed
cases of cerebral malaria, were largely devoid of significant intravascular change.
These patients, together with those in the group of patients with non-malarial comatose
illness, illustrate the fact that coma may accompany infections with extensive systemic
inflammation but without evidence of intracerebral inflammatory responses. It is,
therefore, possible that, in the CM(B) and CM(C) patients (with cerebral sequestration
and cerebral intravascular HO-1 induction), the observed widespread systemic inflammation
may have been similarly important in the pathogenesis of coma. This adds strength
to the argument that systemic events, not only those in the brain, should be borne
in mind when attempting to understand disease outcome in African children clinically
diagnosed with cerebral malaria. The CM(A) group, one third of the cases, in which
malaria was only one of several conditions that could have contributed to death, might
have provided insight into the state the cerebral vasculature of the CM(B) and CM(C)
cases after they had become comatose, but some time before death.

Finally, the inhibitory activity of CO, the product of HO-1, against TNF [4] parallels that of NO, the product of iNOS [5]. Ideas on how iNOS polymorphisms might have been selected for in African populations
[36,37] through their interaction with malarial disease might also, from these results, apply
to known HO-1 polymorphisms [38,39] in these populations.

Acknowledgments

This work was supported by the Australian National Health and Medical Research Council,
Grant 148902, the National Institute of Allergy and Infectious Diseases, USA (Grant
1-RO1-AI 34969) and the Wellcome Trust. We thank Dr Biziwick Mwale, Director of the
Queen Elizabeth Central Hospital, and Professor Robin Broadhead, Department of Paediatrics
of the College of Medicine, University of Malawi, for their hospitality and for access
to patients in the Malaria Ward, and we thank Professor Terrie Taylor, who played
a major part in running the Severe Malaria Ward and in arranging and conducting autopsies.
We appreciate the cooperation of parents and guardians of the children studied, and
the devoted care provided by the nursing and laboratory staff within the Paediatric
Department and the research programme. This study received funding from the Australian
Health and Medical Research Council, the National Institutes of Health, USA, and The
Wellcome Trust, UK.